A Functional Study of an Orphan Nuclear Receptor Estrogen-Related Receptor alpha in Prostate Cancer

Background and aims of study: Prostate cancer is the most common cancer in many Western counties among the male populations. Latest cancer statistics also show that its incidence and mortality rates are rapidly increasing in China and Hong Kong (Prostate cancer ranked the 3rd common cancer and 5th cancer causing death in Hong Kong in 2009). Current therapeutic strategies of prostate cancer mainly target to the antagonizing androgen signaling pathway, which usually drives the disease to the impasse of castration resistance albeit the side effects caused by the imbalance of hormone. Given the hypoxic microenvironment of prostate cancer and the energy regulatory role of ERRalpha, it is hypothesized that ERRalpha might play an active role in the cellular hypoxic adaptation of prostate cancer hence advancing the progression of this disease.;Materials and methods: To investigate the functional significance of ERRalpha in cellular hypoxic adaptation of prostate cancer, the following experimental approaches were employed and performed in my thesis study: 1) to survey the expression pattern of ERRalpha in human prostate cancer tissues by immunohistochemical staining; 2) to generate ERRalpha-stable expressing cell lines in selected prostate cancer cell lines and functionally characterize their in vitro phenotypes under normoxia condition; 3) to characterize in vitro hypoxic-response phenotypes of ERRalpha-infectants; 4) to determine the tumorigenicity of ERRalpha-infectants in immuno-deficient SCID mice and to investigate their tumor angiogenesis by immunohistochemical staining; 5) to determine the HIF-1alpha signal cohort in ERRalpha-infectants by both RT-PCR and immuno blot analysis and to investigate the transactivation effect of ERRalpha on HIF-1 targeting genes promoters by dual luciferase reporter assay; 6) to further characterize the hypoxic adaptation phenotypes induced by ERRalpha transduction using shRNA-mediated gene knockdown approach; 7) to further elucidate the effect of ERRalpha on the hypoxic cell growth regulation of prostate cancer by treating ERRalpha-infectants with an ERRalpha-selective antagonist XCT790; 8) to further investigate the mechanisms via which ERRalpha interferes with the protein expression or stabilization of HIF-1alpha as well as HIF-1 signal cohort using immuno blot analysis, immunoprecipitation assays and fluorescence resonance energy transfer (FRET) analysis.;Results: My results are briefly summarized as follows: 1) ERRalpha exhibited an increased immuno expression pattern in high-grade prostate cancer; 2) Ectopic expression of ERRalpha in LNCaP prostate cancer cell line could promote its in vitro cell proliferation, clonal formation, cell-extracellular matrix attachment and cell invasion capacities under both normoxic and hypoxic conditions; 3) Ectopic expression of ERRalpha in LNCaP prostate cancer cell line could promote its in vivo tumorigenicity and tumor angiogenesis; 4) Overexpression of ERRalpha could up-regulate protein level of hypoxia regulatory transcriptional factor-1(HIF-1) alpha subunit (HIF1-alpha) and enhance its transcriptional activity; 5) mRNA knock-down of ERRalpha could attenuate in vitro cell growth capacity of LNCaP prostate cancer cell line under hypoxic condition; 6) Treatment with an ERRalpha specific antagonist XCT790 could inhibit in vitro hypoxic cell growth of LNCaP cells via its effect on decreasing the protein level of HIF-1alpha; 7) ERRalpha could physically interact with HIF-1alpha and such ERRalpha-HIF1-alpha interaction might help to inhibit protein degradation of HIF-1alpha.;Conclusion: The results obtained in this study indicated that ERRalpha could promote the hypoxic cell growth of prostate cancer via its enhancing the protein level of HIF-1alpha and activation of HIF-1 signal cohort. Both treatment with ERRalpha selective antagonist and down-regulating of ERRalpha by shRNA-mediated gene knockdown approach could attenuate the hypoxia adaptation of prostate cancer cells, which might be mediated by their suppression of the protein level of HIF1alpha. ERRalpha could directly interact with HIF-1alpha and such interaction might help to suppress the protein degradation of HIF1alpha, suggesting that ERRalpha may play an active role in hypoxic adaptation in advancing of prostate cancer. (Abstract shortened by UMI.).